Method of verifying the synthesis of organic molecules using nuclear magnetic resonance spectroscopy

ABSTRACT

A NMR method to verify the presence of organic molecular compounds consisting of repetitive occurring individual structures is presented. The method comprises the steps of assigning structure codes to the selected compounds, in accordance with the respective starting compounds used, measuring multi-dimensional NMR spectra from at least some of the compounds, uniquely assigning signal groups of NMR spectra to the individual structures, checking the NMR spectra of the compounds for the presence of all assigned signal groups, and characterizing a particular compound as being TRUE if the check of its particular combination of structures yields the result that the signal groups of structures contained in its total code had been observed. The method permits rapid and accurate verification of the presence of compounds having repetitive structures such as those produced in combinatorial chemistry.

This application is a continuation of Ser. No. 09/888,596 filed on Jun.26, 2001 now abandoned which is a continuation in part of Ser. No.09/422,639 filed Oct. 22, 1999 now abandoned and claims Paris ConventionPriority of DE 198 49 231.6 filed Oct. 26, 1998 the complete disclosureof which are all hereby incorporated by reference.

BACKGROUND OF THE INVENTION

The invention relates to a method of verifying the synthesis of organicmolecules using nuclear magnetic resonance spectroscopy. The method isparticularly suited for use in libraries of compounds produced bycombinatorial chemistry.

A large number of new organic compounds can be automatically synthesizedfrom a smaller number of molecular structures using the techniques ofcombinatorial chemistry. These molecular structures contributing to theproduct are assigned a molecular structure code. Methods have beenproposed for the subsequent verification of the success or failure ofthe synthesis (see for example “COMBINATORIAL” by A. W. Czarnik,Analytical Chemistry News & Features, pages 378 A to 386 A, 1 Jun.1998).

Combinatorial chemistry methods aim at synthesizing compounds using asmall number of chemical reactants in all combinations defined by agiven reaction scheme to obtain a large number of well-defined products.NMR methods can be used to verify synthesis of these products with highthroughput. The assessment of the measured NMR spectra has beenconventionally carried out “manually” and mainly intuitively by highlyspecialized chemists and has also been based on relatively inaccuratemodel calculations.

The purity control and structure verification of compound librariesproduced by automated synthesis and combinatorial chemistry both play anessential role in the success of medicinal chemistry programs. Highperformance liquid chromatography (HPLC), mass spectrometry (MS) andliquid chromatography-mass spectrometry (LC-MS) techniques are generallyaccepted as the most appropriate means of characterization. Althoughthese analytical methods are fast and easy to automate, they do notprovide sufficient structural and quantitative data about the desiredproducts.

Nuclear magnetic resonance (NMR) spectroscopy is the most informativeanalytical technique and is widely applied in combinatorial chemistry.However, an automated interpretation of the NMR spectral results isdifficult. The interpretation can usually be supported by use ofspectrum calculation and structure generator programs. Automatedstructure validation methods rely on ¹³C NMR signal comparison usingmolecular structure/molecular-spectra correlated databases or shiftprediction methods.

In view of these aspects of prior art, it is the object of the presentinvention to present an NMR method which permits rapid, reproducible andreliable verification of a large number of molecular compounds producedby combinatorial chemistry.

SUMMARY OF THE INVENTION

This object is achieved with a nuclear magnetic resonance (NMR) methodfor verifying a production of compounds within a library of organiccompounds produced by combinatorial chemistry, wherein the organiccompounds are generated by reacting a first class of first molecularstructures with at least one additional second class of second molecularstructures, the compounds in the library being prepared having knownfirst and second molecular structure content, wherein a first commonstructure class designation and first individual structure indexdesignations are assigned to each of the first molecular structures anda second common structure class designation and second individualstructure index designations are assigned to each of the secondmolecular structures the method comprising the steps of:

-   -   a) selecting a subset of the library compounds which contains        all of the first molecular structures and all of the second        molecular structures;    -   b) measuring a multi-dimensional NMR spectrum of each individual        compound in said subset;    -   c) adding and subtracting NMR spectra of individual compounds in        said subset to generate a combined NMR spectrum for each of said        first and said second molecular structures, each of said        combined NMR spectra having enhanced intensity contributions        from one of said first and said second structures;    -   d) uniquely assigning a signal group in said combined NMR        spectra to individual ones of said first and said second        molecular structures and to said associated first and second        structure classes and indices;    -   e) measuring a multi-dimensional NMR spectra of a sample which        may contain an organic compound of the library other than those        organic compounds used in the compound subset;    -   f) examining said NMR spectra taken in step e) for a presence of        all uniquely assigned signal groups;    -   g) if said examining of a particular NMR spectrum indicates a        presence of first and second molecular structures from said        first and said second class corresponding to a particular        organic compound of the library, characterizing that compound as        TRUE; and    -   h) repeating steps e) through g) on differing samples until all        desired organic compounds in the library have been examined.

The sequence of the method steps is preferably carried out in theabove-mentioned order, but may proceed in a reasonably modifieddifferent order. For example, correlation of signal groups in the NMRspectra to individual structures may be effected on the basis ofpreviously obtained information, even prior to step a).

The advantages of the inventive method are now illustrated with anexample of a three component reaction. Such a three component reactioninvolves linking three classes of molecule structures (building blocks)A, B, C to form a product compound denoted ABC. Each class may containseveral molecular structures (a₁, A₂ . . . A_(i); B₁, B₂ . . . B_(i);C₁, C₂ . . . C_(i)). With just 10 molecular structures in each class,1000 different product compounds can be formed. Thus, the structures inthe synthesized product compounds can be formally represented as acombination of individual molecular structures with one structure comingfrom each class. In many cases, a non-variable region (core) occurs inall library compounds. A structure code A_(x)B_(y)C_(z) defined by thesynthesis can be assigned to each product component. Both spectroscopicand chromatographic data can be regarded as the sum of data belonging tothe individual molecular structures of a compound molecule.

Since synthesized product compounds can be formally represented as acombination of individual molecular structure fragments, 2D NMR spectracan be regarded as the sum of spectra of these structures. Throughsystematic examination of e.g. 2D C,H correlated NMR spectra theindividual contributions of each molecular structure to the spectrum canbe isolated into sub-spectra.

Once the spectral patterns of all individual molecular structures havebeen defined, all available spectra can be tested for the presence of aparticular structure in the synthesized compounds. The proposedstructure is verified (true) if all expected molecular structurefragments are found. If at least one of the expected patterns is notfound, then the spectrum is not verified (false). Spectra with a lowsignal-to-noise ratio, or with large amounts of impurities areautomatically assigned a “vague” category. In the simplest case, theverification procedure can be based on the integration of spectralpatterns and comparison to an automatically detected noise level. Betterresults are obtained if a signal (e.g., from the core) can be defined asan internal reference signal to normalize all integrals. A referencespectrum is then defined for each molecular structure pattern. Thecorresponding integrals of these reference spectrum are defined as 100%and corresponding integral values of all other spectra are re-scaledaccordingly. During the verification it is then possible to apply anadditional threshold which expresses the minimum signal intensity ofidentified patterns. For example, a spectrum related to the structurecode A₁B₁C₁ would be classified as true if A₁, B₁, and C₁ are identifiedand at least each integral exceeds 30%.

In a preferred variant of the inventive method, if a check of aparticular compound yields the result that the signal group of at leastone molecular structure contained in the compound molecule was notobserved in the NMR spectra, this compound is characterized “false”. Inthis manner, proper synthesis according to plan is characterized as“true” and those compounds where the synthesis did not work out, (atleast not completely) are recognized through the absence of at least oneof the molecular structure signal groups in the NMR spectrum.

In a further improvement, the NMR spectra are examined for a signal tonoise ratio and/or a core signal intensity and a combination ofmolecular structures is characterized as “vague” if the signal to noiseratio or the core signal intensity is less than a certain thresholdvalue.

The classification of “vague” is generally given when too littlesubstance was available in the sample for the measuring time, leading topoor signal to noise ratios. For spectra exhibiting a core, “vague”results can be associated with core signal intensities which are below acertain threshold value.

Since the subset of individual compounds is generally considerablysmaller than the entire library of all possible combinations, thechecking of the NMR spectra for the remaining combinations can beconsiderably accelerated. Clearly, the prerequisite therefore is that aunique assignment of each molecular structure contained in the compoundsubset to a signal group is actually possible. If this should not be thecase, the subset has to be augmented and a new attempt for uniqueassessment must be pursued. Only when the assignment is unique, can allremaining NMR spectra be checked for the signal groups determined withthe assistance of the subset to verify synthesis of the remainingcompounds in the library.

A further embodiment of a preferred variant of the method ischaracterized in that the subset is derived through modification of asubset of compounds used in a previous measurement series. Usingprevious results and assignments as a guide, a new subset of compoundscan be selected which is more likely to meet the criterion of a uniqueassignment of signal groups to the individual molecular structures.

The number of compounds in the subset is preferably minimized bygrouping molecular structures into classes having identical or similarchemical behavior. The smallest possible number of compounds in thesubset is equal to the number of molecular structures in the largestclass.

The assignment of signal groups in the NMR spectra of the subsetcompounds to the individual molecular structures can also be carried outmanually. In this case, no special assessment software is required.Alternatively, the assignment is preferably carried out automaticallyvia computer, which is considerably faster than “manual assessment”.Costly personnel are not required for the actual assessment and theverification is carried out objectively, in a reproducible fashion, andlargely error-free.

In a further preferred variant of the inventive method, the assignmentof signal groups to the molecular structures is carried out using NMRspectra from a previous measurement series and/or on the basis oftheoretically calculated spectral data. Using preexisting information,the assessment can be accelerated by using a selected subset ofstructure combinations and the assignment of signal groups to thestructures can be carried out directly.

The organic molecules to be synthesized are preferably of low molecularweight, in a molecular weight range of approximately 100 u toapproximately 2000 u. This is a mass range which is preferred incombinatorial chemistry. There are sufficient numbers of molecularstructures in this mass range for carrying out the NMR measurements.Furthermore, two-dimensional NMR spectra are still relatively easy toanalyze in this molecular weight range.

The synthesized organic compound molecules preferably contain a sectionreferred to as a “core” present in all molecules of the library whichcan be consequently characterized in the NMR spectra by a common signalgroup and used as an internal reference for normalizing intensities. Thecore may be added prior to synthesis as an independent reactant, whereinthe other structures couple thereto. Alternatively, a core can be formedin a coupling range of the initial molecular structures themselves, as acommon section of the compound.

The core is preferable a molecular structure having between two and sixchemical coupling points. In this case, the possible number ofcombinations remains sufficiently clear. Moreover, a multitude ofcommercial substances can be used as a core with this kind of coresub-structure.

In a particularly preferred further development of the method, themolecular structures in a class of structures are assigned to arespective common coupling point of the core.

The number of structures should be considerably greater than three tomake a combinatorial approach reasonable at all.

The NMR spectra are preferably two-dimensional, 13C/1H correlatedspectra (e. g. HSQC spectra=hetero nuclear single quantum coherence, seee.g. J.Magn.Reson. B108, pages 94–98 (1995)). Two-dimensional NMRspectra can be generated in rather short measuring times on the order ofminutes with a resolution which is substantially better than that ofone-dimensional spectra only. The multi-dimensional NMR spectrumpreferably comprises signals stemming from coupling between ¹H and ¹³Cnuclei. In this way, the two most important atomic species of organicchemistry are included.

The assignment of signal groups in the NMR spectra to the individualmolecular structures can be carried out particularly easily by formaladdition and subtraction of normalized spectra of the associatedstructure codes. In a computer automated application of the method, thecorresponding data can be quickly processed in this manner with theassistance of cluster algorithms.

In order to reduce the information content of the NMR spectra to theessential relevant features, a further preferred variant of the methodprovides peak lists which are established from the multi-dimensional NMRspectra to define the signal groups.

In a particularly easy standard for the recognition of peaks, a datapoint of the multi-dimensional, preferably two-dimensional, NMR spectrumis recognized as a “peak” if its value is larger than those of the nneighboring data points, wherein e.g. 4≦n≦12, preferably n=8.

In a preferred further development, neighboring peaks are combined intoclusters and are assessed by means of cluster analysis, wherein one ormore clusters are assigned to a given molecular structure as a signalgroup. In this manner, a two-dimensional definition of the signal groupsis possible. This method is insensitive to the fine structure of theindividual peaks, which can be neglected. The analysis of the cluster assuch is described e.g. in K. -P. Neidig et al., Journal of MagneticResonance 89, pages 543 to 552 (1990).

In a particularly preferred further development, the method assigns acluster surface to each cluster in the two-dimensional NMR spectrum(more generally: a hyper surface in a multi-dimensional spectrum) and amolecular structure is regarded as recognized if, for all clustersurfaces assigned thereto, the NMR signal integrated over the clustersurface exceeds a predetermined threshold value. In this way, a highlyreliable pattern recognition of molecular structures in the NMR spectrumis possible.

The threshold value can be defined as a normalized constant. Thethreshold value is preferably chosen normalized to the integral of theNMR signal over cluster surfaces assigned to other structures. Thisenables normalization of the respective signal group and is particularlyuseful for a core molecular structure. The threshold value is thenindependent of the absolute intensities of the spectra.

In a further preferred variant of the method, a table is established todisplay the results of analysis of NMR spectra measured for theremaining compounds in the library, recording the molecular structures(columns) and whether they were recognized (+) or not (−). In threeadditional columns, the sum of the recognized molecular structures, atotal assessment (“true” or “false”, possibly “vague”) and the requiredcombined molecular structure code is indicated. In this manner, thetotal result of the combinatorial measuring series can be convenientlysummarized.

Further advantages of the invention can be derived from the descriptionand the drawing. The features mentioned above and below can be usedindividually or in any arbitrary combination. The embodiments shown anddescribed are not to be understood as exhaustive enumeration but ratherhave exemplary character for illustrating the invention.

The invention is shown in the drawing and is further explained by meansof an embodiment.

BRIEF DESCRIPTION OF THE DRAWING

FIG. 1 a shows a schematic representation of an organic molecularcompound formed from the molecular structures A_(X)+B_(Y)+C_(Z) whereinthe structures form a common core;

FIG. 1 b shows a schematic representation of an organic molecularcompound formed from the structures A_(X)+B_(Y)+C_(Z) and a coremolecule;

FIG. 2 shows the structures used in the spectra of FIGS. 3 to 7

FIGS. 3 through 7 each show an NMR spectrum of a combination of threestructures A_(X), B_(Y), C_(Z) with a core molecule, namely

FIG. 3 A₂+B₂+C₂;

FIG. 4 A₂+B₁+C₁;

FIG. 5 A₂+B₁+C₃;

FIG. 6 A₂+B₂+C₃;

FIG. 7 A₁+B₂+C₂;

FIG. 8 shows a second example of the invention, having a library ofninety-six 4-phenylbenzopyrans generated in a three component reaction;

FIG. 9 shows how linear combinations of spectra can be used to extractpattern box C₃;

FIG. 10 shows decomposition of a 2D HSQC spectrum of a compound intosubspectra corresponding to each of the three molecular structurefragments A₂, B₁, and C₁;

FIG. 11 shows a 1D spectrum of a synthesis product;

FIG. 12 illustrates synthesis of 4-phenylbenzopyran library 1; and

FIG. 13 illustrates results of automated NMR analysis in accordance withthe invention in comparison to ESIMS, and HPCL analysis.

DESCRIPTION OF THE PREFERRED EMBODIMENT

Chemical compounds of a combinatorial series are particularly useful forautomatic or at least partially automatic interpretation, since thesignals of the structures in the combinatorial series can be separatedformally into a core module, which is identical for all members of theseries, and into a few variable module classes which are variedsystematically via a limited number of structural fragment species inthe class.

FIG. 1 a shows such a compound in a highly schematic fashion. Thecombined organic molecule consists of three molecular structures A_(X),B_(Y) and C_(Z) which form a common core section in the range of theirmutual connections (dashed lines in FIG. 1 a).

FIG. 1 b shows an alternative in which the combined organic compound isformed with its own core molecular structure and having three attachedmolecular structures A_(X), B_(Y) and C_(Z). These compound moleculescan be described by structure codes which consist of a structure classwith a corresponding structure index A_(X), B_(Y) and C_(Z) etc. Theindices x, y, z each represent a species or structure and are successiveintegers (1, 2, 3 . . .).

Such structure elements can be identified as signals or signal groups intwo-dimensional HSQC spectra as shown below. The examples shown in FIGS.3 to 7 are chemical substances represented in FIG. 2. Referring to FIG.2 one can define:

(1) 4-nitrophenyl=B2

(2) phenyl=B1

(3) 3,4methylenedioxy-phenoxy=A2

(4) 3-hydroxy,4-bromo-phenoxy=A1

(5) tert-butyloxycarbonyl-piperazyl=C2

(6) morpholinyl=C1

(7) 2-methoxy-piperazyl=C3

The results of NMR experiments are shown in FIGS. 3 to 7. The spectrarepresent two-dimensional so-called HSQC (hetero nuclear single quantumcoherence) experiments. Applied to protons and carbon (13C), the signalsin those spectra show the correlation between carbon atoms and protonschemically bound thereto, i.e. the carbon signals in the direction δ1and the proton signals in the direction δ2.

Identification of signal groups belonging to a certain molecularstructure can be carried out manually or automatically. For automaticanalysis, one performs formal algebraic additions and subtractions onthe spectra associated with specific structure codes to isolate signalsoriginating from a particular structure. For analysis purposes one canassign the value “1” to each structure present in a particularcombination and use a threshold to extract a particular structure.Consider the following structure combinations:

A₂ B₂ C₂

A₂ B₁ C₁

A₂ B₁ C₃

A₂ B₂ C₃

A₁ B₂ C₂

The addition of

A₂ B₁ C₃

A₂ B₂ C₃ and

subtraction of

A₂ B₂ C₂

A₂ B₁ C₁

A₁ B₂ C₂

yields the following sums for the structures

A₁=1

A₂=0

B₁=0

B₂=−1

C₁=−1

C₂=−2

C₃=2

If one sets the threshold value at 2, only C₃ remains.

The general rule is as follows: Add all N structure codes which containthe desired sub-structure, subtract others, and set the threshold valueto <=N (e. g. N/2).

This formal operation can be carried out in practice on peak listsobtained from the corresponding spectra. A data point is therebyrecognized as a two-dimensional peak if its value is larger than each ofits 8 nearest neighbors. Neighboring peaks can be combined by means of acluster analysis which evaluates distances and intensities to formgroups (clusters).

When a peak of a spectrum is added to the peaks of another spectrum, itis included in the associated list with an increase in intensity for allpeaks which are within a pre-defined radius.

When a peak of a spectrum is subtracted from the peaks of anotherspectrum, it is removed from the respective list and the intensities ofall peaks which are within a pre-defined radius are reduced.

The result is a list of peaks which originate from the signals of thedesired structure. Since these signals may be slightly different invarious spectra, the peaks appear several times and in groups. Thegroups or clusters are determined by a cluster analysis.

The signals obtained for the desired structure are represented by smallrectangles in the spectra, with each rectangle containing exactly onecluster. The width and height of these areas correspond to the expectedvariations of the signals in the given set of spectra.

When the remaining spectra are checked, integration of all structures iscarried out. (Summation of all corresponding data points). Furthermore,for each spectrum, a pre-defined area which does not contain any signalsis integrated and a noise value is calculated therefrom. The noise valueis subtracted from all integrals.

Signals of the “core” structure can be defined as a reference andintegrated separately. The integral ratios between all areas of allstructures can also be calculated.

A structure is regarded as recognized if all of its areas have anintegration value >0. A structure can also be regarded as recognized ifall its areas exceed a defined integration value, compared to areference value.

A molecular structure could also be regarded as recognized if all ratiosof the integrals of all of its areas to the integrals of all other areasof all other structures exceed a defined value.

In the embodiment of FIGS. 2 through 7, x=2, y=2 and z=3. This resultsin 2×2×3=12 possible combinatorial combinations (A_(X) B_(Y) C_(Z)). Theminimum subset for correlating the signals of each structure would haveto comprise at least three molecules to assure that C₁, C₂ and C₃ areall present.

The results of the measurements is summarized in the following table:

Combinations A₂B₂C₂ A₂B₁C₁ A₂B₁C₃ A₂B₂C₃ A₁B₂C₂ A₁ − − − − + A₂ + + + +− B₁ − + + − − B₂ + − − + + C₁ − + − − − C₂ + − − − + C₃ − − + + −Core + + + + + Sum 4 4 4 4 4 Result + + + + +

FIGS. 8 to 13 illustrate results for another library.

FIG. 8 shows ninty-six 4-phenylbenzopyrans generated in a threecomponent reaction. For x=6, y=4 and z=4, x×y×z=96 different compoundswith the structure codes A_(x)B_(y)C_(z) are obtained. Library 1 ischaracterized by x+y+z+core=15 different structural fragments and asubset of six of the ninety-six compounds contain all structuralfragments (e.g. A₁B₁C₁, A₂B₂C₂, A₃B₃C₃,A₄B₄C₄, A₅B₁C₃ and A₆B₂C₄).

FIG. 9 illustrates a linear combination of spectra to extract patternbox C₃. Signals are peak picked and transformed into peak areas.Overlapping peak areas of spectra containing the structural fragmentcode C₃ are added (counted) and peak areas of spectra not containing C₃are subtracted. The threshold is adjusted so that only peak areas of C₃remain, and after a clustering step, boxes are defined for eachremaining peak area.

FIG. 10 illustrates decomposition of a 2D HSQC spectrum of a compoundinto sub-spectra corresponding to each of the three molecular structurefragments A₂, B₁, and C₁. The width and height of the boxes indicate theexpected range of chemical shift for the signals of a given fragment. Aspectral pattern is defined by the combination of the correspondingboxes. In FIG. 10A the spectral patterns of each fragment are found andthe structure of the expected compound A₂B₁C₁ is therefore validated. InFIG. 10B the structure of compound A₂B₁C₁ is not verified because thespectral patterns of both A₂ and C₁ are missing.

FIG. 11 shows a 1D spectrum of a synthesis product. Different signalsare related to different molecular fragments.

FIG. 12 illustrates synthesis of 4-phenylbenzopyran library 1.

FIG. 13 illustrates results of the automated NMR method of the inventionin comparison to an ESIMS, and HPLC analysis. Each cell contains theexpected structure code, the final assignment, and the data for NMR (topleft), ESIMS (top middle), and HPLC (top right). Light gray colorationmeans that the proposed structure is “true” in NMR, gives the expectedmolecular ion in ESIMS, and shows the expected retention time in HPLC.Dark grey means that the proposed structure is “false” following NMR,does not give a diagnostic molecular ion in ESIMS, or the retention timediffers from the expected one. White is given for “vague” results inboth NMR and ESIMS. HPLC purity is given in % (top right). Combinedresults are given in the structure code field (light grey: “true”, darkgrey: “false”, white: “vague”). The classification “true” of the HPLCanalysis was not taken into consideration for the final assignment.Contradictory results lead to the final category “vague”. Eighteencompounds were not obtained by the synthesis procedure (B10, C1, C6,C12, D1, D3, D4, D8, D9, E12, F12, G1, G9, G11, H1, H7, H8, H11).

The 4-phenylbenzopyran library 1 was synthesized using a multi-componentreaction by the combination of phenols, unsaturated aldehydes andsecondary amines (FIG. 12). The products were purified before analysis.The ¹H NMR and 2D HSQC spectra of the ninety-six 4-phenyl-benzopyranswere measured using standard NMR probes (5-mm) within sixteen hours.

The software analysis of the spectra includes the following steps:

1. Enter into the software:

-   -   a) list of codes for the possible molecular structure fragments        involved in the combinatorial reaction    -   b) construct the paths to the recorded spectra and the        associated structure codes.

2. Perform calculation step to define the integration boxes for eachmolecular structure fragment. The outputs are boxes assigned to eachfragment.

3. Perform calculation step to determine appropriate reference spectra.

4. Perform calculation step to integrate all spectra. As an output agraphical display in table format, optionally using three colors (red,green, and yellow) to characterize the samples is shown and a textualresult list is written on disk.

The following table summarizes verification results for the example ofFIGS. 8 through 13.

NMR result A₁ A₂ A₃ A₄ A₅ A₆ B₁ B₂ B₃ B₄ C₁ C₂ C₃ C₄ A₁B₁C₁ + + − − − −− + − − − + − − − A₁B₂C₁ + + − − − − − − + − − + − − − A₁B₃C₁ − + − − −− − − − − − + − − − A₁B₄C₁ ? + − − − − − − − − + − − − − A₁B₁C₂ + + − −− − − + − − − − + − − A₁B₂C₂ + + − − − − − − + − − − + − − A₁B₃C₂ − + −− − − − − + − − − + − − A₁B₄C₂ − + − − − − − − − − + − − − − A₂B₁C₁ +− + − − − − + − − − + − − − A₂B₂C₁ + − + − − − − − + − − + − − −

In the columns labeled with fragment codes, the “+” and “−” entriesindicate whether or not the corresponding spectral pattern wasidentified in a given spectrum. The column labeled NMR results indicateswhether the structure is verified (+), false (−) or vague (?). Forexample, for compound A₁B₃C₂ pattern A₁, B₂, and C₂ were identified andthe compound was assigned false. In this case the sample has beenexchanged and the correct structure code would be A₁B₂C₂.

1. A nuclear magnetic resonance (NMR) method for verifying a productionof compounds within a library of organic compounds produced bycombinatorial chemistry, the organic compounds generated by reacting afirst class of first molecular structures with at least one additionalsecond class of second molecular structures, with the compounds in thelibrary being prepared having known first and second molecular structurecontent, wherein a first common structure class designation and firstindividual structure index designations are assigned to each of saidfirst molecular structures and a second common structure classdesignation and second individual structure index designations areassigned to each of said second molecular structures, the methodcomprising the steps of: a) selecting a subset of library compoundswhich contains all of said first molecular structures and all of saidsecond molecular structures; b) executing a two dimensional NMR pulsesequence to generate, after completion of the pulse sequence, a twodimensional NMR spectrum of each individual compound in said subset; c)adding and subtracting linear combinations of the NMR spectra resultingfrom step b) of individual compounds in said subset to generate anindividual combined NMR spectrum for each of said first and said secondmolecular structures, each of said individual combined NMR spectrahaving enhanced intensity contributions from only one of said first andsaid second structures; d) uniquely assigning a signal group in saidcombined NMR spectra to individual ones of said first and said secondmolecular structures and to the associated first and second structureclasses and indices; e) executing a two dimensional NMR pulse sequenceto generate, upon completion of the pulse sequence, two dimensionalspectra of a sample to examine an organic compound of the library otherthan those organic compounds used in the compound subset; f) analyzingsaid NMR spectra taken in step e) for a presence of all uniquelyassigned signal groups; g) if said analyzing of a particular NMRspectrum indicates a presence of first and second molecular structuresfrom the first and said second class corresponding to a particularorganic compound of the library, characterizing that compound as TRUE;and h) repeating steps e) through g) on differing samples until alldesired organic compounds in the library have been examined.
 2. Themethod of claim 1, wherein should step g) indicate that at least one ofsaid signal groups of said first and said second molecular structures ofa particular organic compound has not been observed, that particularcompound is characterized to be FALSE.
 3. The method of claim 2, furthercomprising the step of examining, prior to steps g) and h), said NMRspectra for at least one of a signal to noise ratio and a core signalintensity and characterizing a compound as VAGUE if at least one of saidsignal to noise ratio and said core signal intensity is less than athreshold value.
 4. The method of claim 1, further comprising expandingsaid subset of compounds to include additional individual compounds ifsteps a) to d) indicate failed synthesis of one or more desiredindividual compounds.
 5. The method of claim 1, wherein the organiccompounds have molecular weights in a range from 100 u to 2000 u.
 6. Themethod of claim 2, wherein the organic compounds have molecular weightsin the range from 100 u to 2000 u.
 7. The method of claim 1, wherein acommon core is present in all compounds.
 8. The method of claim 3,wherein a common core is present in all compounds.
 9. The method ofclaim 7, wherein said core is a molecular structure with between 2 and 6binding sites.
 10. The method of claim 1, wherein a number of first andsecond molecular structures is between 5 and
 500. 11. The method ofclaim 1, wherein said two dimensional NMR spectrum is a two-dimensional¹H and ¹³C correlated spectrum.
 12. The method of claim 1, wherein saidassignment of signal groups in said combined NMR spectra to individualmolecular structures is achieved by formal addition and subtraction ofnormalized structure codes.
 13. The method of claim 1, wherein aspectral region of said two dimensional NMR spectrum is recognized instep g) to be a peak if its intensity is larger than that of nneighboring equal sized data regions, wherein 4≦n≦12.
 14. The method ofclaim 13, wherein neighboring recognized peaks are combined intoclusters and are analyzed by means of cluster analysis, wherein one ormore clusters are assigned to certain molecular structures as saidsignal group.
 15. The method of claim 14, wherein a cluster area isassigned to each cluster inside a two-dimensional NMR spectrum andwherein a particular molecular structure is recognized to be identifiedif an NMR signal, integrated over said cluster area, is greater than apredetermined limit for all cluster areas assigned to said particularmolecular structure.
 16. The method of claim 15, wherein said limit is aratio to an integral of NMR signal over cluster areas which are assignedto other molecular structures.
 17. The method of claim 1, wherein thelibrary comprises three classes of molecular structures.
 18. The methodof claim 8, wherein said core is used as an internal reference fornormalizing intensities.
 19. The method of claim 1, wherein said twodimensional NMR spectra are heteronuclear single quantum coherencespectra.